Skip Navigation

Cardiovascular Research 2004 63(4):689-699; doi:10.1016/j.cardiores.2004.04.020
© 2004 by European Society of Cardiology
This Article
Right arrow Abstract Freely available
Right arrow FREE Full Text (PDF) Freely available
Right arrow E-letters: Submit a response
Right arrow Alert me when this article is cited
Right arrow Alert me when E-letters are posted
Right arrow Alert me if a correction is posted
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Add to My Personal Archive
Right arrow Download to citation manager
Right arrowRequest Permissions
Right arrow Disclaimer
Google Scholar
Right arrow Articles by Wakisaka, Y.
Right arrow Articles by Izumi, T.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Wakisaka, Y.
Right arrow Articles by Izumi, T.
Social Bookmarking
Add to CiteULike   Add to Connotea   Add to Del.icio.us  
What's this?

Copyright © 2004, European Society of Cardiology

Structural and electrical ventricular remodeling in rat acute myocarditis and subsequent heart failure

Yuko Wakisaka*, Shinichi Niwano, Hiroe Niwano, Junko Saito, Tohru Yoshida, Shoji Hirasawa, Hideaki Kawada and Tohru Izumi

Department of Internal Medicine, Kitasato University School of Medicine, 1-15-1, Kitasato, Sagamihara, 228-8111, Japan

* Corresponding author. Tel.: +81-42-778-8111; fax: +81-42-778-8441. Email address: wakisaka{at}med.kitasato-u.ac.jp

Received 1 February 2004; revised 18 April 2004; accepted 20 April 2004


   Abstract
Top
 Abstract
1. Introduction
 2. Methods
 3. Results
 4. Discussion
 References
 
Objective: We reported that experimental autoimmune myocarditis (EAM) rats showed dramatic changes in ventricular action potential and enhanced arrhythmogenicity in the acute phase, but mechanisms for this are still unclear. To investigate the mechanisms of cardiac remodeling in acute myocarditis and subsequent heart failure, physiological and molecular changes were evaluated along the time course of EAM. Methods: Six-week-old Lewis rats were immunized with porcine cardiac myosin. On days 14, 21, 35 and 60 after immunization, histology, hemodynamics and electrophysiological parameters (i.e., effective refractory period (ERP), monophasic action potential duration (MAPD) and PVC inducibility) were evaluated and compared with control rats. After these studies, the expression levels of Kv+ and L-Ca2+ channels, ion transporters and BNP expressions in the left ventricle were examined by quantitative real time RT-PCR and Western blot analysis. Results: EAM rats showed acute myocarditis with massive infiltration of the mononuclear cells on days 14 and 21. Subsequently, a chronic dilated cardiomyopathy (DCM)-like structural change was observed on day 60. Hemodynamic parameters were worse in EAM than controls. ERP and MAPD were longer in EAM than controls, with a peak on day 21, which was parallel to PVC inducibility. mRNA levels of Kv4.2, Kv1.5, KChIP2, frequenin and SERCA2a, and the protein levels of Kv4.2 and Kv1.5, were reduced, especially in the acute phase. Conclusions: The initial reduction of Ito-related molecules, such as the expression levels of Kv4.2, 1.5, frequenin and KChIP2, and the prolongation of MAPD are considered to be a key mechanism of ventricular remodeling and cause the characteristic clinical findings in EAM in the acute inflammatory phase and chronic DCM phase.

KEYWORDS Remodelling; Ventricular arrhythmias; Myocarditis; Ion channels; Cardiomyopathy


   1. Introduction
Top
 Abstract
 1. Introduction
2. Methods
 3. Results
 4. Discussion
 References
 
Electrical remodeling can be observed in various heart diseases, such as ventricular tachycardia [1,2], ischemia [3–5], cardiomyopathy [6,7] and congestive heart failure [6,7], which appear with the progression of various types of structural remodeling. In spite of these different conditions, the main body of electrical remodeling is commonly prolongation of the action potential duration caused through reductions in transient outward K+(Ito) currents in animal models although the role of down-regulation of KCNH2 was reported in humans. Electrical remodeling is one of the results of structural remodeling, but a recent report by Sah et al. showed ventricular hypertrophy caused by the intrinsic reduction of Ito in a Kv4.2N transgenic mouse model [8,9]. It was suggested that electrical remodeling itself can influence structural remodeling. Therefore, the mechanisms of the progression of electrical remodeling must be understood along with its interaction with structural remodeling.

Experimental autoimmune myocarditis (EAM) in the rat is a unique and useful model for understanding giant cell myocarditis and subsequent dilated cardiomyopathy (DCM) [10]. Since EAM rats show dramatic changes in the characteristics of cardiac conditions during its time course, it is an interesting model for evaluating the interactions between electrical and structural remodeling [11,12]. We previously reported on electrical remodeling in the acute phase characterized by prolongation of ERP and monophasic action potential duration (MAPD) and increased arrhythmogenicity [13]. However, these characteristics might change during the chronic phase because the cardiac structure, pathological findings and environments of the myocardium mediated by cytokines' expressions, etc., are varied. To clarify the mechanisms of ventricular remodeling in the EAM rat, the hemodynamics, electrophysiological properties and molecular bases such as expression levels of ion channels or exchanger-related molecules were analyzed in the myocardium.


   2. Methods
Top
 Abstract
 1. Introduction
 2. Methods
3. Results
 4. Discussion
 References
 
2.1. Preparation and immunization
Six-week-old male Lewis rats were immunized with purified cardiac myosin in each rear footpad as previously described [13]. The control rats received injections of 0.25 ml of saline in the same manner. All studies were performed in accordance with the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health (NIH Publication No. 85-23, revised 1996) and Ethics Committee of the Kitasato University School of Medicine.

2.2. Electrophysiological evaluation
On each day among days 14, 21, 35 and 60 after the initial immunization, the heart was exposed by a median sternotomy under interperitoneal anesthesia with 50 mg/ml sodium pentobarbital (1 ml/kg body weight for controls and 0.5 ml/kg for EAM groups). The same system was used for the electrophysiological evaluation as previously described [13]. A pair of platinum needle electrodes (0.1 mm) was used for electrical stimulation and recording. The analogue signals were converted into digital signals at a sampling frequency of 1000 Hz (Power Lab 8sp, Bio Research, Tokyo, Japan) and stored on a computer hard disk. The band pass filter was set at 50–300 Hz for standard cardiac electrogram recording and at open –300 Hz for recording of the monophasic action potential (MAP). To evaluate the ventricular effective refractory period (ERP) and the inducibility of the ventricular arrhythmias, 2-ms step shortening of the coupling interval of the extrastimulus was employed in two basic cycle lengths of 150 and 120 ms. The inducibility of the ventricular arrhythmias was calculated as a ratio between the time of induced arrhythmias and the time of attempts for arrhythmia induction. The MAP duration (MAPD) was determined as the interval between the onset of the MAP trace and 20% (MAPD20) and 90% repolarization times (MAPD90) [13].

2.3. Hemodynamic parameters
For the hemodynamic parameters, left ventricular systolic pressure (LVSP) and left ventricular end-diastolic pressure (LVEDP) were monitored by a needle tip micromanometer (SPR477, Millar, USA), which was directly inserted into the left ventricle through the ventricular apex. Positive and negative dP/dt was calculated with a single differentiator (G46 15-71, Gould, USA).

2.4. Heart weight and histology
After electrophysiological measurement and before sampling the ventricular muscle, the weight of the whole heart was measured and the ratio of the heart and body weight (HW/BW) was calculated. In randomly selected rats, the heart was transversely sliced and fixed in 10% formalin, embedded in parafilm and stained with hematoxylin–eosin or Azan–Mallory for histological evaluation.

2.5. Plasmid construction and quantitative real time RT-PCR
Total RNA was prepared from the left ventricular free wall as previously described [13] and was treated with DNase I (Stratagene, CA, USA). cDNA was synthesized from 3 µg of total RNA with reverse transcriptase (Invitrogen, CA, USA) in a final volume of 20 µl. The mRNA levels of the voltage dependent K+ channels (Kv1.4, 4.2, 4.3, 1.5 and erg), Ito-regulating molecules (K+ channel-interacting protein-2; KChIP2 and neuronal calcium center protein-1; frequenin), cardiac ion transporters (sarcoplasmic reticulum Ca2+-ATPase 2a; SERCA2a, ryanodine receptor; RyR and Na+–Ca2+ exchanger; NCX), L-type Ca2+ channel and BNP were evaluated by quantitative RT-PCR. As a specific internal control for the myocyte, the level of troponin T mRNA was also evaluated [13]. The standard plasmid for the real-time RT-PCR was prepared as follows; RT-PCR was performed using each primer's pair (Table 1) and the amplified DNA was inserted into the pGEM-T vector (Promega, WI, USA). The recombinant plasmid was isolated after transforming it into JM109 competent cells using a Plasmid Mini-prep Kit (Qiagen, CA, USA). The plasmid was used as the standard sample after the sequence identities were confirmed.


View this table:
[in this window]
[in a new window]

  		Table 1 PCR primers used for amplification of ion channels and cardiac transporters genes

 
Real-time PCR was performed with QuantiTectTM SYBR Green PCR Master Mix (Qiagen) using an ABI PRISM 7700 Sequence Detection System (PE Biosystems, NJ, USA). The PCR reactions were cycled 50 times by a three-step cycle procedure (denaturation 95 °C, 15 s; annealing 58 °C, 1 min; extension 72 °C, 1 min) after the initial stages (50 °C, 2 min; 95 °C, 10 min). To make the standard curve, serially diluted standard plasmids were analyzed at the same time. An ABI PRISM 7700 System calculated a standard curve, and the absolute copy numbers of 14 species of mRNA in the samples were obtained.

2.6. Western blot analysis
Cardiac tissues were homogenized in ice cold lysis buffer containing 50 mM Tris–HCl (pH 7.4), 50 mM EDTA, 150 mM NaCl, 0.1% Triton X-100, 10 µg/ml aprotinin, 200 mM PMSF and 1 mg/ml leupeptin. The homogenates were mixed with loading buffer (50 mM Tris–HCl, 100 mM dithiothreitol, 2% SDS, 0.1% bromophenol blue, 10% glycerol) and solubilized at 95 °C for 10 min. For Western blotting, 50 µg of total protein were separated by 10% SDS–polyacrylamide gel electrophoresis and electroblotted to a PVDF transfer membrane (Amersham Pharmacia Biotech, UK). After blocking with 5% nonfat milk, the membrane was incubated with anti-Kv4.2, anti-Kv1.5 antibody (CHEMICON International) or anti-troponin T antibody (JLT12; Sigma, USA) and subsequently with the second antibody (goat anti-rabbit IgG antibody or rabbit anti-mouse IgG antibody) conjugated to alkaline phosphatase. After the development with 5-bromo-4-chloro-3-indolylphosphate and nitro blue tetrazolium chloride (BCIP/NBT), the densities of each band in the digitized images were measured using the public domain NIH Image program.

2.7. Numbers of rat in each study protocol
In total, 28 rats were immunized in this study and autoimmune myocarditis was induced in all rats. Because 5 rats died suddenly during acute phase, the remaining 23 were used for the evaluation, i.e., 6 for each day of 14, 21 and 60, and 5 for day 35. As the control, 15 rats (3 for each day of 0, 14, 21, 35 and 60), which underwent saline injection, were used. For the presentation, the controls for electrophysiological and hemodynamic data were shown separately, but the controls for mRNA and protein data were represented by day 0 because there was no difference among the days of 0, 14, 21, 35 and 60.

2.8. Statistical analysis
All quantitative data are described as mean±S.E.M. Basic comparative statistics were performed using an unpaired Student's t test and p<0.05 was considered to be statistically significant.


   3. Results
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
4. Discussion
 References
 
3.1. Histopathology
Macroscopically, the hearts were markedly enlarged and the ventricular wall thickness was increased in EAM. The surface of the heart was discolored, especially on days 14 and 21, indicating acute myocarditis. Microscopically, large numbers of mononuclear cells, including giant cells, had infiltrated the epicardial layer of the ventricular wall. On day 35, the ventricular wall thickness was still elevated in comparison with the control, but the number of inflammatory cells was reduced and many fibroblasts appeared in the intestinum. On day 60, the ventricle was then dilated and the wall thickness was rather thin. There were dense collagen fibers surrounding the surviving myocytes and the inflammatory cells had disappeared (Fig. 1).


Figure 1
View larger version (89K):
[in this window]
[in a new window]
[Download PowerPoint slide]
  		Fig. 1 Cross-sections of a control heart on day 21 and EAM hearts on days 14, 21, 35 and 60. Left panels show the cross-sections at low magnification (40 x, hematoxylin/eosin or Azan/Mallory staining). Areas indicated by the arrows in the left panels are magnified (100 x) in the right panels. The EAM heart was enlarged due to infiltration of inflammatory cells and edema in the early days and large numbers of mononuclear cells were observed in the myocardium (B, C). During the later days, the number of inflammatory cells was decreased (D, E) and massive fibrosis was observed in the intestinum (E). See text for discussion.

 
3.2. Heart weight and hemodynamic parameters
Table 2 represents the data of HW/BW and the hemodynamic parameters. HW/BW was always higher in EAM rats than the controls, showing its peak on day 14. Although HW/BW gradually reduced along the time course, it never returned to the level found in the control rats even on day 60. LVEDP was higher on days 14 and 60 in EAM than the controls. The +dP/dt was lower at all time points especially on day 60, and the –dP/dt was also lower at all points except on day 14.


View this table:
[in this window]
[in a new window]

  		Table 2 Homodynamic characterization control and EAM

 
3.3. Electrophysiological evaluation
Along the time course, ventricular ERP was prolonged in EAM once in its acute phase, but recovered in the chronic phase (Fig. 2A). The difference was significant on days 14, 21 and 35 for both BCL of 150 and 120 ms. The degree of ERP prolongation was largest on day 21 for a basic cycle length of 120 ms (82.0±10.5 vs. 55.0±2.1 ms; p<0.001) and 150 ms (85.6±20.9 vs. 54.7±3.6 ms; p=0.0037) compared with the control. Fig. 2B shows representative examples of recording traces of the ventricular MAP recorded at the left ventricular free wall in control and EAM rats. In the control rats, the initial depolarization formed a shape peak and repolarization occurred immediately after the depolarization peak. In contrast, the EAM rats showed a prolonged MAP shape, and the initial depolarization peak was followed by a relatively slow repolarization phase causing a wide dome. MAPD20 was longer in EAM rats than the controls on day 21 for BCL of 150 ms and on days 14, 21 and 60 for BCL of 120 ms. MAPD90 was always longer in EAM than controls, maximally on day 21 (BCL=150 ms: 113.1±11.8 vs. 69.4±15.1 ms; p=0.0004, BCL=120 ms: 110.2±39.1 vs. 62.2±3.8 ms; p=0.024) (Fig. 2C).


Figure 2
Figure 2
View larger version (40K):
[in this window]
[in a new window]
[Download PowerPoint slide]
  		Fig. 2 Panel A: The changes in ventricular ERPs in EAM (n=6 on days 14, 21 and 60, n=5 on day 35) and control rats (n=3 for each day). The ventricular ERPs were once prolonged in EAM during the acute phase, but recovered during the chronic phase. Data are described as mean±S.E.M. *p<0.05, **p<0.01 vs. control. Panel B: Representative traces of the ventricular MAP in the control on day 21 and EAM on days 14, 21, 35 and 60. In the control, the initial depolarization formed a sharp peak which was followed by immediate repolarization. In contrast, the EAM showed prolonged MAP, and the initial peak was followed by a relatively slow repolarization phase causing a wide dome. Panel C: Changes in MAPD in the two groups. MAPD20 and MAPD90 were longer in the EAM group (n=6 on days 14, 21 and 60, n=5 on day 35) than in the control group (n=3 for each day), maximally on day 21. Data are described as mean±S.E.M. See text for discussion. *p<0.05, **p<0.01 vs. control.

 
Ventricular arrhythmias (i.e., PVC or VT) were induced by ventricular single extrastimuli totally in 13/23 EAM rats (56.5%) and in 3/12 controls (25.0%). In EAM rats, the incidence of PVC induction was significantly higher than in controls maximally on day 21 (16.7% vs. 3.47%, p=0.008). Although the difference in PVC inducibility became non-significant during the chronic phase, PVC inducibility under isoproterenol infusion (0.05 µg/kg/min) was still higher in EAM than controls (45.4% vs. 24.0%, p=0.035, Fig. 3).


Figure 3
View larger version (24K):
[in this window]
[in a new window]
[Download PowerPoint slide]
  		Fig. 3 Inducibility of PVC or VT by single premature stimulus. The inducibility was higher in EAM rats (n=6 on days 14, 21 and 60, n=5 on day 35) than controls (n=3 for each day), especially during the acute phase. Numbers in parentheses indicate total attempts for PVC induction in each group. Although the PVC inducibility became lower during the chronic phase, it was still higher under ISP infusion (0.05 µg/kg/min) in EAM rats than controls. See text for discussion.

 
3.4. Expression of cardiac ion channels, their regulators, transporters and BNP
The absolute copy numbers of 14 species of mRNA in the samples are shown in Table 3. The expression level of cyclophilin mRNA, which is commonly used as an internal control [4,21], was elevated in EAM maximally during the acute phase. This increase is suspected to be the result of the influence of a number of infiltrated heterogeneous cells, because it was reported that other housekeeping genes (i.e., G6PDH and β-actin) were also elevated in the heart of the same EAM model [14]. In contrast, troponin-T is highly specific to myocytes [15,16] and the expression level of troponin-T mRNA was depressed in EAM during the acute phase. Since this decrease was compatible to the relative decrease in ventricular myocytes in a given amount of ventricular tissue, the expression level of troponin-T mRNA was used as an internal control.


View this table:
[in this window]
[in a new window]

  		Table 3 Absolute copy numbers of 14 mRNA/15 ng total RNA of control and EAM

 
The absolute copy numbers of Kv4.3, erg, L-Ca2+, RyR, NCX and frequenin mRNA were reduced in EAM rats compared with the controls, but the difference was not significant after the division by copy numbers of troponin-T (data was not shown). The expression levels of Kv4.2, 1.5, KChIP2 and SERCA2a were depressed in EAM, especially in the early phase, even after correction by troponin-T expression levels (Fig. 4). The expression level of BNP mRNA was elevated in EAM during the acute and chronic phases.


Figure 4
View larger version (20K):
[in this window]
[in a new window]
[Download PowerPoint slide]
  		Fig. 4 Relative copy numbers of cardiac potassium channels and cardiac transporters determined by real-time RT-PCR with internal control of troponin-T. Results were calculated by the ABI PRISM 7700 System using standard curves for the positive control plasmid, and are expressed at the means±S.E.M. The expression levels of Kv4.2, 1.5, KChIP2 and SERCA2a were significantly depressed in EAM (n=6 on days 14, 21 and 60, n=5 on day 35) especially during the early phase. See text for discussion. *p<0.05, **p<0.01 vs. control (n=3 on day 0).

 
The level of Kv4.2 protein was significantly reduced in EAM than the control on day 21 even after the correction by troponin-T level, but recovered during the chronic phase. The level of Kv1.5 protein was also significantly reduced on day 21 after the correction by troponin-T level (Fig. 5). These results coincided well with change in the mRNA levels of Kv4.2 and 1.5.


Figure 5
View larger version (22K):
[in this window]
[in a new window]
[Download PowerPoint slide]
  		Fig. 5 Western blot analysis of Kv4.2, 1.5 and troponin-T protein levels. Panel A shows membrane proteins of Kv4.2, 1.5 and troponin-T. The Kv4.2 protein band was detected as the 66-kDa band, and so be Kv1.5 as the 75-kDa and troponin-T as the 38-kDa bands. Panel B shows the quantification of each band. The densities of the bands in the digitized images were measured using the public domain NIH Image program. The protein levels of Kv4.2 and 1.5 corrected by the level of troponin-T were significantly reduced in EAM (n=6 on days 14, 21 and 60, n=5 on day 35) than the control (n=3 on day 0) on day 21. See text for discussion. **p<0.01 vs. control.

 

   4. Discussion
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
 4. Discussion
References
 
EAM showed acute myocarditis in its earlier phases characterized by the infiltration of inflammatory cells and inter-cellular edema, and a DCM-like condition in its chronic phase characterized by fibrosis and ventricular dilatation. In the present study, we investigated structural and electrical remodeling including its molecular basis in an EAM model and documented the following findings. First, hemodynamic parameters such as LVEDP and left ventricular dP/dt were worse in EAM rats than controls along the time course, but it was most prominent during the acute phase. Second, ventricular arrhythmogenicity became higher in EAM during the acute phase. Although the inducibility of ventricular arrhythmia became lower during the chronic phase, there was still an inducible arrhythmia under isoproterenol infusion. Third, ventricular ERP and MAPD were prolonged in EAM especially during the acute phase. Finally, the expression levels of Kv4.2, 1.5, KChIP2 and SERCA2a mRNA were reduced in EAM during the acute phase but that of Kv4.2 and KChIP2 were recovered during the chronic phase.

4.1. Electrical remodeling and arrhythmogenicity in EAM
In our previous report, we documented an increase in ventricular arrhythmogenicity and the prolongation of ERP and MAPD in acute phase EAM [13]. Although this could be defined as ventricular electrical remodeling in acute myocarditis, the precise mechanisms during the time course was unclear. In the present study, we precisely characterized these changes including changes during the chronic phase.

Overall, ventricular arrhythmogenicity was higher and ERP and MAPD were longer in EAM than in controls throughout the study. However, they were more prominent during the acute phase than the chronic phase, so that ventricular arrhythmogenicity seemed to be parallel to changes in ERP and MAPD. Since the histological findings were so different between the acute and chronic phases, mechanisms of the ventricular arrhythmia might have been different. During the acute phase, triggered activity may play an important role because prolonged action potentials may attribute to inducing an early or delayed after potential [13]. In our previous report, delayed after depolarization was considered to be one of the causes of the induction of ventricular arrhythmia because there was a direct relationship between coupling intervals of the premature stimulus and induced ventricular arrhythmia [13]. In contrast, reentry may play a main role in the induction of ventricular arrhythmia during the chronic phase. Since inter-cellular fibrosis appears during the chronic phase, the cellular coupling might be broken and then a reentrant rhythm might appear. The higher inducibility of ventricular arrhythmia under isoproterenol infusion in EAM during chronic phase is compatible with this mechanism. Overall, electrical remodeling in EAM was not simple but varied depending on the phase of the time course.

4.2. Molecular basis of the electrical remodeling
The major change in MAP trace caused by electrical remodeling in EAM was the "dome formation" during phase 2 of the action potential. This suggests that the change in the ion-current in this electrical remodeling appears relatively early in the action potential and the reduction of early outward current may contribute to this change.

Cardiac Ca2+-independent transient outward current (Ito) is a major component of the outward current, especially in the early phase of the action potential. Ito is encoded by Kv4.2, 4.3 and 1.4 genes, and the down-regulation of these genes results in Ito reduction and the APD prolongation [8,17,18]. A reduction in Kv4.2 and 4.3 gene expression has been documented under various conditions such as ventricular tachycardia [1,2], ischemia [3–5] or cardiomyopathy [6,7] in animal models and humans [19], which was accompanied by APD prolongation. The expression of Kv4 channels is also regulated by various channel-interacting proteins such as KChIP2 [20–22] and frequenin [23,24]. KChIP2 may regulate the expression level and physiological characteristics of Kv4 molecules [22,25,26], and frequenin may enhance Kv4.2 currents, particularly in immature heart [24]. In the present study, the expression levels of Kv4.2, KChIP2 and frequenin mRNA were all depressed in EAM, and these changes were compatible to the change in the MAP (i.e., the dome formation in phase 2). Therefore, the reduction of these Ito-related molecules is considered to be one of the initial molecular changes for introducing electrical remodeling in this model. Recent report showed ventricular hypertrophy caused by an intrinsic reduction of Kv4.2 in the mouse model [8,9,29,34]. The primary reductions of Ito-related molecules may play a role in causing structural remodeling to continue to the chronic phase in the present EAM model.

Kv1.5, which generates a sustained outward current, Iss [27,28] in the rat ventricle, was also depressed in EAM. Although the role of Iss is unclear in the rat ventricle, the reduction of Kv1.5 is compatible with APD prolongation and may contribute to electrical remodeling. The change in L-Ca2+ channel expression is controversial in various heart diseases [29,30]. In this study, although the expression of L-Ca2+ mRNA corrected by troponin-T was unchanged, the expression of SERCA2a mRNA was depressed during the acute phase. The expression level of SERCA2a is depressed in heart failure [31]. Recently, SERCA2a has been considered to be an important factor in initiating cardiac contraction and relaxation in animal models and humans [31,32]. The reduction in SERCA2a expression reduces Ca2+ uptake into sarcoplastic reticulum resulting in intracellular Ca2+-overload. Although it was reported that the reduction in SERCA2a expression in heart failure is due to the reduction of phosphorylation levels of phospholamban [33], there is a range of views regarding this question.

The most interesting finding in this model was the difference between acute and chronic phases. Although the prolongation in ERP and MAPD recovered somewhat during later phases, they were still longer than in the control. Similarly, significant reductions in the expression of Kv1.5, erg, SERCA2a, RyR, NCX and frequenin were still observed during the chronic phase, whereas the expression of Kv4.2, 4.3, L-Ca2+ and KChIP2 recovered during the chronic phase. This indicates that the molecular basis of electrical remodeling is different during acute and chronic phases.

4.3. Signals which introduce the electrical remodeling in EAM
As documented in the previous studies, EAM showed acute myocarditis during acute phase and DCM-like findings during chronic phase [13]. The acute phase was characterized by inflammation (i.e., the infiltration of inflammatory cells and inter-cellular edema), and it was preceded by a dramatic increase in inflammatory cytokines such as TNF-{alpha}, IFN-{gamma}, IL-2, etc. [11,12]. The chronic phase was characterized by inter-cellular fibrosis and ventricular dilatation. Although ventricular wall thickness became even wider during the acute phase, the hypertrophy of myocytes was unclear. Although the wall stress and hypertrophy of cardiomyocytes cause APD prolongation [19,34–36], we could not detect exact differences between the acute and chronic phases. Severe ventricular wall thickening was observed during the acute phase, but hypertrophy of each myocyte was not obvious in comparison with the chronic phase. Since the major difference between the acute and chronic phases in this model was the presence or absence of inflammation, the inflammatory process itself might be considered to contribute to the introduction of electrical remodeling in this model. Our recent report also suggests that inflammatory cytokines may induce a reduction in Kv4.2 expression in the cultured cardiomyocyte. Because Ito reduction and cellular hypertrophy can affect on each other through inter-cellular Ca2+ overload, the electrical and structural remodeling in EAM might be regulated by inflammatory cytokines especially during its acute phase. In contrast, the main finding during the chronic phase was increased wall stress during the diastolic phase, so that the mechanism of electrical remodeling might be similar to other experimental models [19,35,36].

4.4. Clinical implications
This study was the first systematical analysis and characterization of electrical remodeling in an EAM model which has a perfect counter part to clinical cases [10,37]. Acute myocarditis in human shows various types of arrhythmia especially during the acute phase, which was completely compatible to results of this study. Therefore, understanding the mechanism of arrhythmogenicity might be a clue for helping to select an appropriate therapy for arrhythmia in those cases. More importantly, this study is the first report which states the importance of inflammation as the initiation of electrical remodeling. It was reported that increased cytokine levels in the myocardium could be observed in various heart diseases such as ischemic heart disease, cardiomyopathy and heart failures of nonspecific origin [2–4,6,7]. According to the results of the present study, microscopic inflammation may produce an arrhythmogenic focus that may result in arrhythmia under various conditions. This novel understanding of the cause of arrhythmia might lead to the concept that anti-inflammatory therapy can be used for the treatment of arrhythmias in an appropriate state.

4.5. Study limitations
There are a few limitations in this study. First, because the electrophysiological and hemodynamic parameters were evaluated under open-chest anesthetized state, each parameter can be influenced by anesthesia. Importance of induced arrhythmia in this study was unclear, but the fact that 5/28 rats died suddenly during the acute phase may indicate the importance of arrhythmia in this model. Second, because this study used in vivo model, the importance of specific signals to initiate ventricular remodeling could not be separated. This point should be solved in future experiments utilizing cultured myocytes, etc.

4.6. Conclusions
EAM showed ventricular remodeling characterized by a higher ventricular arrhythmogenicity and prolonged ERP and MAPD. The degree of electrical remodeling was more prominent during the acute phase than during the chronic phase. The reduction in expression of mRNAs of Ito-related molecules is considered to contribute to the introduction of electrical remodeling.


   Acknowledgements
 
This study was supported by a grant for scientific research from the Ministry of Education Science and Culture of Japan (No. 11838015), and also by a grant to the Research Committee for Epidemiology and Etiology of Idiopathic Cardiomyopathy from the Ministry of Health and Welfare of Japan.


   Notes
 
Time for primary review 15 days


   References
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
 4. Discussion
 References
 

  1. Yu H., McKinnon D., Dixon J.E., et al. Transient outward current Ito1, is altered in cardiac memory. Circulation (1999) 99:1898–1905.[Abstract/Free Full Text]
  2. Kaab S., Nuss H.B., Chiamvimonvat N., et al. Ionic mechanism of action potential prolongation in ventricular myocytes from dogs with pacing-induced heart failure. Circ. Res. (1996) 78:262–273.[Abstract/Free Full Text]
  3. Qin D., Zhang Z.H., Caref E.B., Boutjdir M., Jain P., El-Sherif N. Cellular and ionic basis of arrhythmia in postinfarction remodeled ventricular myocardium. Circ. Res. (1996) 79:461–473.[Abstract/Free Full Text]
  4. Huang B., Qin D., El-Sherif N. Spatial alterations of Kv channels expression and K+ currents in post-MI remodeled rat heart. Cardiovasc. Res. (2001) 52:246–254.[Abstract/Free Full Text]
  5. Pierre B., Pierre S., Rene C., et al. Markedly different effects on ventricular remodeling result in a decrease in inducibility of ventricular arrhythmias. J. Am. Coll. Cardiol. (1994) 23:505–513.[Abstract]
  6. Thuringer D., Coulombe A., Deroubaix E., Coraboeuf E., Mercadier J.J. Depressed transient outward current density in ventricular myocytes from cardiomyopathic Syrian hamsters of different ages. J. Mol. Cell. Cardiol. (1996) 28:387–401.[CrossRef][Web of Science][Medline]
  7. Thuringer D., Deroubaix E., Coulombe A., Coraboeuf E., Mercadier J.J. Ionic basis of the action potential prolongation in ventricular myocytes from Syrian hamsters with dilated cardiomyopathy. Cardiovasc. Res. (1996) 31:747–757.[CrossRef][Web of Science][Medline]
  8. Wickenden A.D., Lee P., Sah R., Huang Q., Fishman G.I., Backx P.H. Targeted expression of a dominant-negative K(v)4.2 K(+) channel subunit in the mouse heart. Circ. Res. (1999) 85:1067–1076.[Abstract/Free Full Text]
  9. Sah R., Oudit G.Y., Nguyen T.T., et al. Inhibition of calcineurin and sarsolemmal Ca2+ influx protects cardiac morphology and ventricular function in Kv4.2N transgenic mice. Circulation (2002) 105:1850–1856.[Abstract/Free Full Text]
  10. Kodama M., Matsumoto Y., Fujiwara M., et al. A novel experimental model of giant cell myocarditis induced in rats by immunization with cardiac myosin farction. Clin. Immunol. Immunopathol. (1990) 57:250–262.[CrossRef][Web of Science][Medline]
  11. Okura Y., Yamamoto T., Goto S., et al. Characterization of cytokine and iNOS mRNA expression in sutu during the course of experimental autoimmune myocarditis in rats. J. Mol. Cell Cardiol. (1997) 29:491–502.[CrossRef][Web of Science][Medline]
  12. Goren N., Leiros C.P., Sterin-Borda L., Borda E. Nitric oxide synthase in experimental autoimmune myocarditis dysfunction. J. Mol. Cell. Cardiol. (1998) 30:2467–2474.[CrossRef][Web of Science][Medline]
  13. Saito J., Niwano S., Niwano H., et al. Electrical remodeling of the ventricular myocardium in myocarditis—Studies of rat experimental autoimmune myocarditis. Circ. J. (2002) 66:97–103.[CrossRef][Web of Science][Medline]
  14. Hanawa H., Abe A., Hayashi M., et al. Time course of gene expression in rat experimental autoimmune myocarditis. Clin. Sci. (2002) 103:623–632.[Web of Science][Medline]
  15. Voss E.M., Sharkey S.W., Gernert A.E., et al. Human and canine cardiac troponin T and creatine kinase-MB distribution in normal and diseased myocardium: infarct sizing using serum profiles. Arch. Pathol. Lab. Med. (1995) 119:799–806.[Web of Science][Medline]
  16. Ricchiuti V., Sharkey S.W., Murakami M.M., Voss E.M., Apple F.S. Cardiac troponin I and T alterations in dog hearts with myocardial infarction. Am. J. Clin. Pathol. (1998) 110:241–247.[Web of Science][Medline]
  17. Nerbonne J.M. Molecular basis of functional voltage-gated K+ channel diversity in the mammalian myocardium. J. Physiol. (2002) 525(2):285–298.[CrossRef]
  18. Wickenden A.D., Jegla T.J., Kaprielian R., Backx P.H. Regional contribution of Kv1.4, Kv4.2, and Kv4.3 to transient outward K+ current in rat ventricle. Am. J. Physiol. (1999) 276:H1599–H1607.[Web of Science][Medline]
  19. Nabauer M., Kaab S. Potassium channel down-regulation in heart failure. Cardiovasc. Res. (1998) 37:324–334.[Abstract/Free Full Text]
  20. Shibata R., Misonou H., Campomanes C.R., et al. A fundamental role for KChIPs in determining the molecular properties and trafficking of Kv4.2 potassium channels. J. Biol. Chem. (2003) 278:36445–36454.[Abstract/Free Full Text]
  21. Rosati B., Pan Z., Lypen S., et al. Regulation of KChIP2 potassium channel β subunit gene expression underlies the gradient of transient outward current in canine and human ventricle. J. Physiol. (2001) 533:119–125.[Abstract/Free Full Text]
  22. Kobayashi T., Yamada Y., Nagashima M., et al. Contribution of KChIP2 to the developmental increase in transient outward current of rat cardiomyocytes. J. Mol. Cell. Cardiol. (2003) 35:1073–1082.[CrossRef][Web of Science][Medline]
  23. Nakamura T.Y., Pountney D.J., Ozaita A., et al. A role for frequenin, a Ca2+-binding protein, as a regulator of Kv4 K+-currents. J. Biol. Chem. (2001) 98:12808–12813.
  24. Nakamura T.Y., Sturm E., Pountney D.J., et al. Developmental expression of NCS-1 (frequenin), a regulator of Kv4 K+ channels, in mouse heart. Pediatr. Res. (2003) 53:554–557.[CrossRef][Web of Science][Medline]
  25. An W.F., Bowlby M.R., Betty M., et al. Modulation of A-type potassium channels by a family of calcium sensors. Nature (2000) 403:553–556.[CrossRef][Medline]
  26. Kuo H.C., Cheng C.F., Clark R.B., et al. A defect in the Kv channel-interacting protein 2 (KChIP2) gene leads to a complete loss of Ito and confers susceptibility to ventricular tachycardia. Cell (2001) 107:801–813.[CrossRef][Web of Science][Medline]
  27. Casis O., Iriarte M., Gallego M., Sanchez-Chapula J.A. Differences in regional distribution of K+ current densities in rat ventricle. Life Sci. (1998) 63:391–400.[CrossRef][Web of Science][Medline]
  28. Nishiyama A., Kambe F., Kamiya K., et al. Effects of stress on Kv1.5 K+ channel gene expression in the left ventricle of rat hearts. Environ. Med. (1995) 39:141–144.[Medline]
  29. Bodi I., Muth J.N., Hahn H.S., et al. Electrical remodeling in hearts from a calcium-dependent mouse model of hypertrophy and failure. J. Am. Coll. Cardiol. (2003) 41:1611–1622.[Abstract/Free Full Text]
  30. Less H., Shilkrut M., Rubinstein I., Berke G., Binah O. Cardiac dysfunction in murine autoimmune myocarditis. J. Autoimmun. (1999) 12:209–220.[CrossRef][Web of Science][Medline]
  31. Frank K.F., Bolck B., Erdmann E., Schwinger R.H. Sarcoplasmic reticulum Ca2+-ATPase modulates cardiac contraction and relaxation. Cardiovasc. Res. (2003) 57:20–27.[Abstract/Free Full Text]
  32. Muller O.J., Lange M., Rattunde H., et al. Transgenic rat hearts overexpressing SERCA2a show improved contractility under baseline conditions and pressure overload. Cardiovasc. Res. (2003) 59:380–389.[Abstract/Free Full Text]
  33. Schmidt U., Hajjar R.J., Kim C.S., et al. Human heart failure: cAMP stimulation of SR Ca(2+)-ATPase activity and phosphorylation level of phospholamban. Am. J. Physiol. (1999) 277(2 Pt 2):H474–H480.[Web of Science][Medline]
  34. Zobel C., Kassiri Z., Nguyen T.T., et al. Prevention of hypertrophy by overexpression of Kv4.2 in cultured neonatal cardiomyocytes. Circulation (2002) 106:2385–2391.[Abstract/Free Full Text]
  35. Volk T., Nguyen T.H., Schultz J.H., Faulhaber J., Ehmke H. Regional alterations of repolarizing K+ currents among the left ventricular free wall of rats with ascending aortic stenosis. J. Physiol. (2001) 530:443–455.[Abstract/Free Full Text]
  36. Kassiri Z., Zobel C., Nguyen T.T., Molkentin J.D., Backx P.H. Reduction of I(to) causes hypertrophy in neonatal rat ventricular myocytes. Circ. Res. (2002) 90:578–585.[Abstract/Free Full Text]
  37. Stull L.B., DiIulio N.A., Yu M., et al. Alterations in cardiac function and gene expression during autoimmune myocarditis in mice. J. Mol. Cell. Cardiol. (2000) 32:2035–2049.[CrossRef][Web of Science][Medline]

Add to CiteULike CiteULike   Add to Connotea Connotea   Add to Del.icio.us Del.icio.us    What's this?


This article has been cited by other articles:


Home page
 Am. J. Physiol. Heart Circ. Physiol.Home page
M. E. Loewen, Z. Wang, J. Eldstrom, A. Dehghani Zadeh, A. Khurana, D. F. Steele, and D. Fedida
Shared requirement for dynein function and intact microtubule cytoskeleton for normal surface expression of cardiac potassium channels
Am J Physiol Heart Circ Physiol, January 1, 2009; 296(1): H71 - H83.
[Abstract] [Full Text] [PDF]

Home page
 Am. J. Physiol. Heart Circ. Physiol.Home page
P. S. Petkova-Kirova, E. Gursoy, H. Mehdi, C. F. McTiernan, B. London, and G. Salama
Electrical remodeling of cardiac myocytes from mice with heart failure due to the overexpression of tumor necrosis factor-{alpha}
Am J Physiol Heart Circ Physiol, May 1, 2006; 290(5): H2098 - H2107.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow FREE Full Text (PDF) Freely available
Right arrow E-letters: Submit a response
Right arrow Alert me when this article is cited
Right arrow Alert me when E-letters are posted
Right arrow Alert me if a correction is posted
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Add to My Personal Archive
Right arrow Download to citation manager
Right arrowRequest Permissions
Right arrow Disclaimer
Google Scholar
Right arrow Articles by Wakisaka, Y.
Right arrow Articles by Izumi, T.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Wakisaka, Y.
Right arrow Articles by Izumi, T.
Social Bookmarking
Add to CiteULike   Add to Connotea   Add to Del.icio.us  
What's this?